Periodic mechanical stress enhances rat chondrocyte area expansion and migration through Src-PLCgamma1-ERK1/2 signaling.

The signal transduction pathways of chondrocyte area expansion and migration under periodic mechanical stress remain a matter of debate. We explore this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, we find that both rat chondrocyte area and migration are significantly amplified. These changes are associated with increases in the phosphorylation of Src, PLCgamma1 and ERK1/2. Area expansion, migration and phosphorylation of PLCgamma1-Tyr(783) and ERK1/2-Thr(202)/Tyr(204) are inhibited (p<0.05) after pretreatment with Src inhibitor (PP2). We further demonstrate that area expansion, migration and phosphorylation of ERK1/2-Thr(202)/Tyr(204) are significantly inhibited (p<0.05) after pretreatment with PLCgamma1 inhibitor (U73122); the phosphorylation site of Src-Tyr(418) is not affected. After pretreatment with an ERK1/2 inhibitor (PD98059), area expansion and migration are inhibited (p<0.05), while the phosphorylation sites of Src-Tyr(418) and PLCgamma1-Tyr(783) are not affected. These findings suggest that periodic mechanical stress promotes chondrocyte area expansion and migration in part through the Src-PLCgamma1-ERK1/2 signaling pathway.

Nong L ，Yin G ，Ren K ，Tang J ，Fan W ... -  《-》

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1.

Ren K ，Ma Y ，Huang Y ，Liang W ，Liu F ，Wang Q ，Cui W ，Liu Z ，Yin G ，Fan W ... -  《-》

Periodic mechanical stress activates integrinβ1-dependent Src-dependent PLCγ1-independent Rac1 mitogenic signal in rat chondrocytes through ERK1/2.

The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of proliferation and matrix synthesis when chondrocytes were exposed to periodic mechanical stress. We observed that periodic mechanical stress statistically and significantly enhanced the phosphorylation and activation of Rac1 (p<0.05 for each). Pre-treatment with the Rac1 selective inhibitor NSC23766 attenuated periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (p<0.05 for each) and abrogated ERK1/2 signal activation (p<0.05), but did not block periodic mechanical stressinduced Src and PLCγ1 phosphorylation in this context. In addition, inhibition of Src with its selective inhibitor PP2 and shRNA targeted to Src blocked Rac1 signal activation (p<0.05 for each), but inhibition of the activity of PLCγ1 did not affect the phosphorylation and activation levels of Rac1 under conditions of periodic mechanical stress. The up-regulation of proliferation and matrix synthesis was inhibited in chondrocytes in response to periodic mechanical stress after pretreatment with blocking antibody against integrinβ1 (p<0.05 for each) but not after pretreatment with blocking antibody against integrinβ3. The phosphorylation levels of ERK1/2, Rac1, PLCγ1 and Src, and Rac1 activation level were also reduced when integrinβ1 was blocked in this context (p<0.05 for each). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part by activating the ERK1/2 mitogenic signal through the integrinβ1-Src-PLCγ1/Rac1-ERK1/2 pathway, which links these important signaling molecules into mitogenic cascades.

Ren K ，Liu F ，Huang Y ，Liang W ，Cui W ，Wang Q ，Fan W ... -  《-》

Periodic mechanical stress stimulates the FAK mitogenic signal in rat chondrocytes through ERK1/2 activity.

The biological effects of periodic mechanical stress on chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying chondrocyte mechanosensing and signaling in response to periodic mechanical stress remain to be determined. In the current study, we examined the effects of focal adhesion kinase (FAK) signaling on periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Periodic mechanical stress significantly induced sustained phosphorylation of FAK at Tyr(397) and Tyr(576/577). Reduction of FAK with targeted shRNA via transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutant Y397F or Y576F-Y577F abolished periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis, accompanied by attenuated ERK1/2 phosphorylation. However, activation of Src, PLCγ1 and Rac1 was not prevented upon FAK suppression. Furthermore, pretreatment with the Src-selective inhibitor, PP2, and shRNA targeted to Src or suppression of Rac1 with its selective inhibitor, NSC23766, blocked FAK phosphorylation at Tyr,(576/577) but not Tyr,(397) under periodic mechanical stress. Interestingly, FAK phosphorylation neither at Tyr(397) nor at Tyr(576/577) was affected by PLCγ1 depletion when periodic mechanical stress was applied. In addition, Tyr(397) and Tyr(576/577) phosphorylation levels were reduced upon pretreatment with a blocking antibody against integrin β1 under conditions of periodic mechanical stress. Our findings collectively suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through at least two pathways, integrin β1-Src-Rac1-FAK(Tyr(576/577))-ERK1/2 and integrin β1-FAK (Tyr(397))-ERK1/2.

Liang W ，Ren K ，Liu F ，Cui W ，Wang Q ，Chen Z ，Fan W ... -  《-》

Periodic Mechanical Stress Induces Extracellular Matrix Expression and Migration of Rat Nucleus Pulposus Cells Through Src-GIT1-ERK1/2 Signaling Pathway.

Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.

Gao G ，Li H ，Huang Y ，Yin J ，Jiang Y ，Xu N ，Zhou D ，Nong L ，Ren K ... -  《-》